Gene DvRPT1 with salt tolerance as well as encoding protein and applications of gene DvRPT1

A technology that encodes proteins and genes, used in applications, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2017-11-07
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no literature reporting that RPT1 has the function of improving biological salt tolerance

Method used

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  • Gene DvRPT1 with salt tolerance as well as encoding protein and applications of gene DvRPT1
  • Gene DvRPT1 with salt tolerance as well as encoding protein and applications of gene DvRPT1
  • Gene DvRPT1 with salt tolerance as well as encoding protein and applications of gene DvRPT1

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Experimental program
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Embodiment 1

[0023] Embodiment one: Dunaliella viridis DvRPT1 Cloning and analysis of the full-length coding region

[0024] Dunaliella viridis D. viridis The EST sequence (Expressed Sequence Tag) of the cDNA library induced by different concentrations of salt was analyzed, and a salt-induced expression sequence (Contig 6774) was found, from which a Dunaliella viridis 26S proteasome regulatory subunit RPT1 gene was isolated and named for DvRPT1 . The full length of the Contig 6774 sequence is 1737 bp, containing DvRPT1 Complete open reading frame (ORF), eukaryotic translation conserved Kozak sequence (gcc atg c) and poly(A) structure (see sequence listing).

Embodiment 2

[0025] Embodiment two: Dunaliella viridis DvRPT1 Encoded protein structure, evolution analysis and location prediction

[0026] The analysis results of Vector NTI software showed that the gene encoded a protein with 433 amino acids, the molecular weight of the protein was 48.3 kDa, and the isoelectric point was 6.8. The encoded protein belongs to the 26S proteasome regulatory subunit RPT1 protein family. The sequence 32-430 AA of DvRPT1 is the conserved domain of RPT1. RPT1 is the regulatory subunit of the 26S proteasome, located in the base structure of the 26S proteasome, and has ATPase activity. The 178-345 AA of the DvRPT1 sequence is the conserved domain of ATP-binding protein, which can combine with ATP to release energy to help deubiquitination of ubiquitination-labeled proteins, thereby entering the base structure of the proteasome to achieve protein degradation.

[0027] figure 1 for the present invention DvRPT1 The evolution analysis diagram of the protein enco...

Embodiment 3

[0029] Embodiment three: Dunaliella viridis DvRPT1 Functional analysis in yeast mutants

[0030] (1) Construction of yeast expression vector

[0031] will first contain DvRPT1 The Contig 6774 sequence of the complete ORF was ligated into a yeast expression vector: We ligated the Contig6774 sequence using the restriction enzyme Eco R I and xho I, construct into yeast expression vector pAJ401 after enzyme digestion, obtain recombinant plasmid pAJ401::6774.

[0032] (2) Transform yeast

[0033]The recombinant plasmid pAJ401::6774 was transformed into yeast salt-sensitive mutant G19 (Mat∂, ura3 his3 trp1 ade2 ena1△::ena4△) using LiAc heat shock transformation method.

[0034] (3) Phenotypic identification of yeast transformants

[0035] The basic medium used is AP medium, and the required amino acids are added. The screening marker was uracil auxotrophy. The vector was transferred to the phenotype display medium of the pAJ401 transformant, and 2% galactose was added as ...

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Abstract

The invention relates to a gene DvRPT1 with salt tolerance as well as an encoding protein and applications of the gene DvRPT1. A DNA sequence of the gene DvRPT1 is shown as SEQ ID NO: 1, and an amino acid sequence of the encoding protein of the gene is shown as SEQ ID NO: 2. The gene DvRPT1 of 26S proteasome regulatory subunit RPT1 (regulatory particle triple-aprotein1) is separated from Dunaliella viridis for the first time. Expression of the gene is induced by salt, and yeast cell transformation proves that the gene has the function of improving salt tolerance of cells and can be used for improving crop characters.

Description

technical field [0001] The present invention relates to genes with salt tolerance function DvRPT1 , its encoded protein and its application. In particular a species of Dunaliella viridis ( Dunaliellaviridis ) 26S proteasome regulatory subunit RPT1 (Regulatory Particle Triple-A protein1) DvRPT1 The nucleotide sequence of the gene, the encoded protein DvRPT1, and its application in improving biological salt tolerance and improving crop traits. Background technique [0002] Dunaliella ( Dunaliella ) is a kind of eukaryotic unicellular green algae without cell wall that widely exists in nature. It is widely distributed in high saline waters such as salt lakes and oceans, and is easy to be isolated from the natural environment. Dunaliella green ( Dunaliella viridis ) is a representative species of Dunaliella. This species is also known as Dunaliella salina, Dunaliella salina, etc., and it is the most salt-tolerant eukaryote known. It has strong adaptability to the envir...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/405C12N15/82
CPCC07K14/405C12N15/8273Y02P20/582
Inventor 李平李艺廉王雪薇唐远平
Owner SHANGHAI UNIV
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